We routinely clone everything in low melting agarose (no purification
whatsoever...) and it works perfectly also with blunts or multiple inserts.
We've found sometimes differences in the quality of different brands and
batches of LMA. The essential step, though, is to use really very competent
bugs (eg. at least 10-9)...
Bruno
moez torki wrote:
> Hi Molbiolers,
>> I often heard that gel purified fragments can be recalcitrant to subsequent
> enzymatic treatments (eg. phosphorylation, ligation, etc..). Although I
> never had problem before, now I am experiencing serious problems in my new
> lab :-( That is I cannot perform even a rudimentary subcloning of my
> TAE-agarose/Geneclean purified fragments. I could get rid of that problem
> by phenol/chloroform/EtOHppt. I could see a whitish pellet and a white
> interface after phenol extraction that I may guess being agarose ? or
> silica ?
> Has anyone bear such troubles ? and is there really significant differences
> in agarose quality (depending on the supplier/price). Indeed I can carry on
> with the phenol extraction but it doesn't really make sense since I am
> using kits to get results faster...
> Thanks for any comment.
>> Moez Torki